Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.
Hepatitis C virus (HCV) is a major public health problem with over 123 million chronic infections estimated worldwide. There is no vaccine or post-exposure prophylaxis available. Currently available therapeutic treatment is limited to administration of ribivirin and pegylated interferon which displays limited efficacy of between 40-80% and causes severe side effects. HCV is the only member of the genus, Hepacivirus within the Flaviviridae family and is grouped into six major genotypes (1-6) and various subtypes (a, b, c, etc.). The high degree of sequence diversity proves a major challenge to the development of a universal vaccine to prevent HCV infection.
HCV encodes two envelope glycoproteins, E1 and E2, present as a heterodimer at the virion surface that mediate viral attachment and fusion to facilitate virus entry. The E1 and E2 glycoproteins are targets for the host immune response, vaccine strategies and the development of antiviral agents.
HCV cellular entry factors include the tetraspanin CD81, scavenger receptor class-B type-I (SR-B1), and the tight-junction membrane proteins Claudin 1, 6, or 9 and Occludin. Several discontinuous CD81-binding motifs have been identified within E2 and are proposed to assemble during folding including polyprotein residue Trp420, Trp437, Leu438, Leu441, Phe442, Tyr577, Trp529, Gly530 and Asp535 as well as amino-acids within the region 613-618. Interactions between the HCV glycoproteins and either Claudin or Occludin have not yet been described although both are essential cofactors for viral entry.
E1 and E2 are type-I transmembrane proteins that are heavily modified during biosynthesis at 4 or 5 and 11 N-linked glycosylation sites, respectively. Expression of E1 and E2 in cis is required for the formation of the functional heterodimer that appears to undergo a slow, cooperative folding pathway facilitated by ER chaperones. Several heterodimerization determinants have been identified within the transmembrane domains of both glycoproteins, the membrane-proximal region of E2 and the W487HY motif within the E2 ectodomain.
Within glycoprotein E2, an independent folding domain (polyprotein residue 384-661) can be efficiently expressed and secreted from cells with the retention of CD8 1 and SR-B1 receptor binding. Located within this receptor-binding domain (RBD; E2661) are three discrete variable regions; the N-terminal hypervariable region 1 (HVR1), HVR2 and the intergenotypic variable region (igVR). Both HVR2 and igVR are flanked by pairs of conserved cysteine residues and all 3 variable regions are believed to be solvent exposed and excluded from the core domain. The E2 RBD is connected to the transmembrane domain (TMD) via a membrane-proximal region containing a conserved heptad-repeat that appears to have structural and functional features analogous to the ‘stem’ region of the flavivirus class II fusion protein glycoprotein E and suggested that E2 may also represent a class II fusion protein.
Glycoproteins E1 and E2 possess 8 and 18 cysteine residues within their respective ectodomains that are conserved across the six major genotypes (FIG. 1A). The arrangement of cysteines and disulfide bonds within these proteins is under investigation, however, it is assumed that they play a role in forming or stabilizing protein folds and therefore play a role in viral binding to host cells, entry into host cells and immunogenicity within the host. Krey et al., PLoS Pathog 6(2): e1000762, 2010 have recently assigned the nine disulfide-bonds formed by these residues within the E2 ectodomain using trypsin proteolysis, redox chemistry and mass spectrometry analysis (Krey et al., 2010 (supra)). The strict conservation of cysteines is indicative of the critical role disulfide bonds play in scaffolding the three-dimensional structure of proteins. Together with secondary structure prediction modeling, Krey et al., 2010 (supra) further proposed a model of E2 as a class II fusion protein; a class of proteins that occur in a number of viruses within the Flaviviridae family (FIG. 1B).
In this class II model of HCV E2, the known CD81-binding regions mapped to the interface of domains I and III. Disulfides 1 and 5 stabilize the domain I β-sheet sandwich while 6, 7 and 8 are located within domain III. The igVR forms a ‘hinge’ between these two domains and disulfides 1 5 and 6. Disulfide 7 was not formally identified in any of the tryptic digests but is assumed to form a disulfide pair. HVR1 is an N-terminal extension external to domain I. Domain II is predicted to form a relatively unstructured domain containing three short-range disulfide pairs: disulfides 2 and 3 flanking HVR2 and disulfide 4 stabilizing the candidate fusion ‘loop’ represented by a sequence of glycine-rich hydrophobic residues between 502-520. Disulfide pair 9 is predicted to lie at the edge of domain III with C677 located within the membrane-proximal or proposed ‘stem’ region of E2.
The formation of disulfide-bonded aggregates and heterogeneous forms of E2 or E1E2 heterodimers when these proteins are recombinantly expressed in a range of expression systems has hindered structural and function studies of HCV. A modified HCV E2 protein that produces conformationally competent polypeptides without these disadvantages is highly sought after.